Project Summary/Abstract Epithelial cells work together to form a protective layer for all the organs they encase yet they turnover through cell death and division at some of the highest rates in the body. To maintain a functional barrier and prevent solid tumors from arising, the numbers of cells that die must match those that divide. We found that mechanical tensions control both processes: when cells are too sparse, stretch rapidly activates cell division, whereas, when cells are too abundant, crowding activates cell extrusion and death. Astonishingly, we identified that a single stretch-activated channel, Piezo1, controls both stretch-induced cell division and crowding-induced cell extrusion. Within only one hour of stretch, Piezo1 activates a population of epithelial cells poised in G2 to accumulate cyclin B and enter mitosis. Conversely, during crowding, Piezo1 activates cells to produce and emit a lipid, Sphingosine 1-Phosphate (S1P), which binds a G-Protein Coupled Receptor in the neighboring cells, S1P2, which activates Rho-mediated actomyosin contraction to squeeze the cell out apically, while maintaining a tight barrier. While apoptotic signaling activates extrusion of dying cells, normally, Piezo1 senses crowding to activate extrusion of live cells that later die by anoikis, or apoptosis due to loss of survival signaling. In this proposal, we investigate how Piezo1 activates cells to divide or extrude and die, depending on the type of force it senses. Piezo1 localizes to the nuclear envelope, ER, and plasma membrane in sparser epithelial regions most likely to divide, and then accumulates into large cytoplasmic plaques in crowded, older cells most likely to extrude. Thus, we propose that Piezo1 can sense stretch in places that need to generate more cells and sense crowding in places that need to eliminate cells through its localization. In our grant renewal, we investigate if Piezo1 levels and localization in sparse versus crowded cells control Ca+2 activation of differential targets to drive cell division or extrusion, respectively. Here we investigate: 1) How does Piezo1 promote extrusion in crowded regions of epithelia? 2) Does calcium activate S1P formation or inactivate adhesion (or other) complexes? 3) How does Piezo1 trigger cell division in sparser epithelial regions? We believe the answers to these questions will give us a better understanding of how cell division and death are governed in epithelia. We also expect our findings to have much broader implications in cancer and other epithelial-based diseases, since we have found that different diseases appear to hijack every facet of extrusion signaling we have identified so far. Thus, uncovering the fundamental pathways that control cell death and division will give us a unique edge is discovering new approaches to treat diseases resulting from their misregulation.